The molecular diagnostics industry has been revolutionized by the advent of nucleic acid amplification technology. Enzyme-based nucleic acid amplification is conventionally practiced using at least two oligonucleotide primers, each primer being complementary to an opposite strand of the nucleic acid that is to be amplified. Also conventionally, the primers are soluble and able to diffuse freely through solution to encounter a complementary target. Upon binding the target, a polymerase enzyme extends the primers using the target nucleic acid sequence as a template for the synthesis of new strands. Detection of the newly synthesized strands provides, either directly or indirectly, a means for detecting the target nucleic acid. As the field has evolved, there has been an emphasis on the development of methods that, in addition to being highly sensitive, are reliable, amenable to automated formats, and minimize the opportunity for operator error.
Various approaches have been used to simplify the manipulations needed to prepare amplification reactions and then carry out amplicon detection steps. For example, reagents used in the amplification step have been freeze-dried so that reconstitution with buffer and addition of a target sample is all that is required to prepare a complete solution-based reaction (see U.S. Pat. No. 5,834,254). In a different approach, self-reporting probes were used in solution-based amplification procedures to avoid the need for separate addition of probe reagents (see U.S. Pat. No. 6,037,130). In yet another instance, electronic biasing was used to direct biotin-labeled amplification primers to discrete areas on a streptavidin-coated microelectronic chip. After immobilizing at those locations, the anchored primers were extended in place by the action of a polymerase using a complementary target sequence as a template, with the extension product being detected following heat denaturation and hybridization of a reporter probe (Westin et al., Nature Biotechnology 18: 199 (2000)). Unfortunately, a stoichiometric relationship between the number of arrayed primers immobilized on the chip and the number of extension products available for detection limits the sensitivity of this latter, microarray-based technique.
The art field generally accepts that microarrays are one of the most promising technologies of the post-genomic era. Simply stated, a microarray is a solid substrate that has molecules immobilized, typically as a grid on its surface. Over 95% of microarray work performed today is carried out using DNA—either cDNA or oligonucleotides—in hybridization formats similar to Southern blotting. While protein microarrays represent an emerging technology, their development has been hampered somewhat because the active conformations of complex proteins have been difficult to preserve through the necessary immobilization procedures. (Gen. Eng. News 21:9 (2001))
The introduction of arraying devices based on inkjet or contact printing technology has greatly simplified laboratory-scale production of microarrays. Indeed, arraying devices for depositing picoliter droplets of solutions onto glass or plastic substrates to create microarrays are now widely available. Rather than synthesizing an oligonucleotide directly on the microarray substrate, these devices allow pre-formed oligonucleotides, or other biological molecules, to be dispensed onto the substrate in a quantity and pattern specified by the user.
One of the challenges underlying efficient production of microarrays relates to the immobilization chemistry that joins the deposited macromolecules to the solid substrate. As indicated above, oligonucleotides have been biotinylated, deposited onto streptavidin-coated surfaces, and immobilized by the noncovalent streptavidin:biotin interaction. While convenient, this approach may not be cost-effective for industrial-scale production. Other approaches based on covalent immobilization chemistries are frequently burdened by complicated synthetic routines that lead to inefficiencies in the yield of desired products.
The present invention addresses the need for nucleic acid amplification techniques that are specific and highly sensitive, and that are compatible with disposable format assays requiring minimal reagent preparation and operator involvement for detection of nucleic acid targets. Also provided is a convenient and versatile covalent immobilization chemistry that may be used for attaching macromolecules to glass and plastic surfaces.